LC Method Development Time Limit

[carlos.teixeira]

Honestly, how many working days do you accept as acceptable for analytical development of a LC method with five analytes, please?

[Peter Apps]

How long is a piece of string ?

Peter

[DR]

Depends on the analytes. If they cover too narrow or too wide a range of polarity and sizes, it may take more than one method or column to get them separated.

If they're of similar size and moderately different polarities, and you ahve been provided with some method development tools and a range of columns to choose from, then it depends on the type of method. Stability indicating methods involve interaction and degradation studies and you also have to incorporate known and potential process impurities. If it's just an assay method, I could probably do it within a week, excluding sample prep optimization (as method development time can also be a function of matrix effects and coming up with sample prep routines to minimize them).

In other words, Peter nailed it. I've knocked single component assays for pharma APIs in a couple of days and I've noted that (once, for me) a known process impurity just couldn't be eluted in a reasonable time frame along with an API and the rest of its related substances, so we went w/ a separate method for that one compound.

[MaryCarson]

Depends on the concentrations you need to analyze, the matrix the analytes are in, and what kind of equipment you have available, etc.

I agree with Peter

[unmgvar]

why 5?
you can spend weeks only on 2 until you get them right.

just for example, we had a case of 14 compounds that we got a method in a day and validated in one week, in another case one compound and one "impurity", took us 15 columns to screen for it and we needed to go over temp. and pH as well. resolution 1.3 at best. about 4 month work on and off, waiting for some of the columns

[lmh]

Oooh, thank you so much for asking this question though! I wish more big chief managers understood just how long it takes to set up a method, and how difficult it is to make such work cost-effective when the final number of samples to be analysed turns out to be only 20-30.

[Russ]

Then there are the times you spend many weeks coming up with a method only to be told that they need to change the formulation and use different raw materials. That is always fun.

[Consumer Products Guy]

Then there are the times you spend many weeks coming up with a method only to be told that they need to change the formulation and use different raw materials. That is always fun.

You mean there are times when that DOESN'T happen ???? Not in my world !!!

We seem to have very tight deadlines, then are told that a fragrance has not yet been chosen. Historically, we have found that the biggest potential interference is from fragrance. For one API, at one time there were at least 30 fragrances used in the various products containing that API, so the API had to be resolved from components in all of them !!!

[Vlad Orlovsky]

With our mixed-mode columns we barely spend 8 hours in developing a method for 5 compounds, in 90% cases it is just few runs if we know structure and our task is defined in terms of sample matrix, detection technique, desire run time and buffer, etc. Mixed-mode allows you to analyze a much wider range of compounds in terms of polarity and ionic properties. These are reasons why we offer free method development for our customers. Just tell your manager that somebody else can do it for free (or don't tell the manager and pretend that you did this work

[Vlad Orlovsky]

why 5?
you can spend weeks only on 2 until you get them right.

just for example, we had a case of 14 compounds that we got a method in a day and validated in one week, in another case one compound and one "impurity", took us 15 columns to screen for it and we needed to go over temp. and pH as well. resolution 1.3 at best. about 4 month work on and off, waiting for some of the columns

I guess you did not start with mixed-mode

[Consumer Products Guy]

Mixed-mode allows you to analyze a much wider range of compounds in terms of polarity and ionic properties. These are reasons why we offer free method development for our customers. Just tell your manager that somebody else can do it for free (or don't tell the manager and pretend that you did this work

My company deems much of such research as trade secret, doesn't like to go outside even if there's a confidentiality agreement, afraid such information will "get out" and tip our hand as to future plans. Believe me, I've inquired about that free method development support several times.

[Russ]

Historically, we have found that the biggest potential interference is from fragrance. For one API, at one time there were at least 30 fragrances used in the various products containing that API, so the API had to be resolved from components in all of them !!!

Agree about the fragrances. More than once I would come up with a method only to have the fragrance changed. As I am sure you are aware, there are often a very large number of peaks seen in any one fragrance which must be separated from the active. At one presentation I had to make to one of the exalted ones in the company, I got some small colored beads from a crafts store and made up 0.03 % (w/w) white beads in black to show what this level really is. It amounted to four white beads in an 8 oz jar almost full of the black ones. I then made up another jar of 0.03 % white beads in a jar containing all the colors of beads of the same size I could find at the crafts store to show what it was really like trying to see a small amount of the active in a formulation containing a large number of separate chemical compunds from all the raw materials in it. For probably the only time I was at that company, they actually seemed to comprehend what I was saying!

[Vlad Orlovsky]

Historically, we have found that the biggest potential interference is from fragrance. For one API, at one time there were at least 30 fragrances used in the various products containing that API, so the API had to be resolved from components in all of them !!!

Agree about the fragrances. More than once I would come up with a method only to have the fragrance changed. As I am sure you are aware, there are often a very large number of peaks seen in any one fragrance which must be separated from the active. At one presentation I had to make to one of the exalted ones in the company, I got some small colored beads from a crafts store and made up 0.03 % (w/w) white beads in black to show what this level really is. It amounted to four white beads in an 8 oz jar almost full of the black ones. I then made up another jar of 0.03 % white beads in a jar containing all the colors of beads of the same size I could find at the crafts store to show what it was really like trying to see a small amount of the active in a formulation containing a large number of separate chemical compunds from all the raw materials in it. For probably the only time I was at that company, they actually seemed to comprehend what I was saying!

This is a nice comparison, but I am not sure that it is a correct one. In your experiment bids are only different in color. In chromatography analytes are different by several parameters (hydrophobicity, polarity, ionic properties, etc). The correct experiment would be to mix colored bids with other shaped colored objects. So even in your case you can separate them by shape and color using simple mesh (after you separate them by color) or just spreading them on a flat surface. Chromatography is science of selectivity.

[unmgvar]

Dear Vlad,
it is just your luck that for that crazy 2 peaks application the 3 mixed modes we screened (1 dionex, 2 from sielc) failed miserably. the final solution was a C-8 column from Zorbax.
the 14 compounds application, were all amine based except ascorbic acid and resorcinol, one, secondary and tertiary. about one third were acids, one third were neutral, one third were bases. a simple high purity silica GP C-18 sepax column with buffer PO4 at pH 3.0 with gradient to ACN was enough. the primesep C we thought would fit for that application was a bust.

[Vlad Orlovsky]

unmgvar,

In 99% cases failure to develop method on mixed-mode related to wrong column or wrong mobile phase (pH, concentration). In your particular case you should go to low pH on Primesep 100 column or pH 4-4.5 on Obleisc R column. You just need to know mechanisms involved and ionization states of each compound and how you control it.

Regarding methods:
If you have mixture of neutral, basic and acidic compounds you have two choices;
1. use tri-modal column which has both cation and anion-exchange groups and also hydrophobic chain. In this case you need to make sure that your compounds are ionized and that stationary phase is ionized. Knowing pKa of compounds and stationary phase will help you to decide on the mobile phase pH
2. use bi-modal column with cation-exchange groups and hydrophobic group. In this case you need to have lower pH and make sure that acidic compounds are not ionized and thus a little bit more hydrophobic. With lower pH you need to make sure that pKa of the column is not above pH of the mobile phase, for example, Primesep C will not retain basic compounds at pH below 3 and at pH above 3, your ascorbic acid will be ionized, but there is no anion-exchange mechanism on Primesep C column.
Once you learn all mechanism mixed-mode will be your primary column. The fact that Dionex, Waters and other companies now have mixed-mode column makes a good statement for the benefits of these column. Problem is that these mixed-mode columns compete with regular RP, ion-exchange columns and column manufacturers might be reluctant to push it in full force.
Mixed-mode is like a sports car with stick shift and 3 pedals instead of automatic transmission and two pedals, it is a little bit more complicated to drive but you will have more fun, will get to place faster, save on gas...and may be pick up some girls on the way . If you don't like SIELC for some reason use other mixed-mode!

Here are few applications:
http://www.sielc.com/Application-Separa ... -HPLC.html

http://www.sielc.com/Application-HPLC-S ... isc-R.html (application similar to yours)

http://www.sielc.com/Application-HPLC-A ... ation.html (application similar to yours)

http://www.sielc.com/Application-HPLC-S ... lumns.html

http://www.sielc.com/Application-Comple ... ounds.html

Don't look at exact structures of compounds in these applications, look from the sand point of properties and mechanisms involved: weak acid, string acid, weak base, strong base, hydrophobic, hydrophilic, etc.
When I talk to people I always say, I don't care what is the exact structure, give me a general description of compounds.
I can challenge anybody with any other column for ANY separation. I need to look at all structures and run 2-3 experiments to get separation and anybody can do the same